Compositions and methods for monitoring oxalate

ABSTRACT

The present invention features compositions and methods for the detection or measurement of oxalate in a sample. Such compositions include test devices that provide for the rapid and accurate detection of oxalate in a sample from a biological fluid. Advantageously, the compositions can be used to monitor the oxalate levels of a patient at a point of care (e.g., at the patient&#39;s home, clinic, physician&#39;s office, or other clinical setting).

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of the following U.S. ProvisionalApplication No. 61/440,133, filed Feb. 7, 2011, the entire contents ofwhich are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Kidney stones are a common disorder of the urinary tract, which arecaused by a buildup of minerals in urine. While many stones passpainlessly through the urinary system, larger stones may causedebilitating pain and may have to be surgically removed. About 1 millionAmericans get kidney stones per year. Some of these individuals have afamily history of kidney stones. The most common type of kidney stonesis formed from calcium oxalate. Patients prone to kidney stone formationare typically placed on a low oxalate diet. However, patient compliancewith dietary restrictions is problematic. Methods for monitoring oxalatelevels in biological samples of a patient throughout the day would allowthe patient to increase water consumption or reduce oxalate consumptionto prevent stone formation. Such methods would also enhance patientcompliance with dietary restrictions by providing almost immediatefeedback on the impact of diet on oxalate levels.

SUMMARY OF THE INVENTION

As described below, the present invention features compositions andmethods suitable for monitoring oxalate levels in a biological sample(e.g., fresh urine sample). Such methods are amenable to use in apatient's home, physician's office, or at any other site where patientcare is carried out. Test devices of the invention provide for the rapidand accurate detection of oxalate levels in a biological fluid (e.g.,urine, blood, plasma).

In one aspect, the invention provides a test device for detectingoxalate in a liquid sample, the device comprising oxalate oxidase, andan indicator system that provides a detectable reaction product inresponse to hydrogen peroxide production. In particular embodiments, theindicator system comprises 3-methyl-2-benzothiazolinone hydrazone (MBTH)and 3-(dimethylamino) benzoic acid (DMAB).

In one aspect, the invention provides a test device for detectingoxalate in a liquid sample, the device comprising oxalate decarboxylase,and an indicator system that provides a detectable reaction product inresponse to formate production. In particular embodiments, the indicatorsystem comprises phenazine methosulfate (PMS) and iononitro tetrazolium(INT).

In one aspect, the invention provides a test device for detectingoxalate in a liquid sample, the device including a liquid permeablematerial defining the following portions in capillary communication: asite for application of a liquid sample, including a liquid permeablemedium; a second portion including a liquid permeable medium; and athird portion that is the site for detecting the presence of oxalate atthe test site, the third portion including a liquid permeable mediumhaving an enzyme system having oxalate specific enzyme activity fixed tothe medium at the test site, the enzyme system including oxalatespecific enzyme, a substance which reacts with a reaction product of theoxalate specific enzyme, and an indicator material which reacts in thepresence of the reaction product and changes color.

In a related aspect, the invention provides a method for detecting ananalyte in a liquid sample, the method involving: applying a liquidsample to a test device according to an aspect of the invention; anddetecting presence or absence of oxalate at a test site, where thepresence of color change at the test site identifies the presence ofoxalate in the sample and the absence of color change at the test siteidentifies the absence of the oxalate in the sample.

In another related aspect, the invention provides a method for detectingoxalate in a liquid sample, the method comprising: applying a liquidsample to a test device according to an aspect of the invention;providing for the flow of the liquid from the site of application to thetest site; and detecting presence or absence of oxalate at a test site,where the presence of color change at the test site identifies thepresence of oxalate in the sample and the absence of color change at thetest site identifies the absence of the oxalate in the sample.

In another aspect, the invention provides a kit containing a reactionmix, the reaction mix including an enzyme system having oxalate specificenzyme activity, the enzyme system including oxalate specific enzyme, asubstance which reacts with a reaction product of the oxalate specificenzyme, and an indicator which reacts in the presence of the reactionproduct and changes color; and a color chart.

In a related aspect, the invention provides a kit comprising a testdevice according to an aspect of the invention.

In still another related aspect, the invention provides an instrumentfor determining oxalate concentrations in a sample, said instrumentincluding: a port to receive a test device according to an aspect of theinvention that includes a component for detecting color change; a lightsource; a detector for detecting reflectance from the support; and adisplay for displaying a result of oxalate presence.

In various embodiments of any of the aspects delineated herein, theenzyme system has oxalate oxidase activity, the enzyme system includingoxalate oxidase, a substance having peroxidative activity, an indicatormaterial which is oxidized in the presence of peroxide, and a substancehaving peroxidative activity and changes color. In various embodimentsof any of the aspects delineated herein, the enzyme system has oxalatedecarboxylase activity, the enzyme system including an oxalatedecarboxylase, a substance having formate dehydrogenase activity, and anindicator material which is reduced in the presence of formate andchanges color.

In various embodiments of any of the aspects delineated herein, the testdevice includes a liquid permeable substrate. In specific embodiments,the liquid permeable substrate is in the form of a strip. In variousembodiments of any of the aspects delineated herein, the device includesoxalate oxidase or oxalate decarboxylase and the indicator system inpowder form, and the device is suitable for containing a liquid.

In specific embodiments, the test device is in the form of a clearliquid container (e.g., cuvette).

In various embodiments of any of the aspects delineated herein, thesecond portion includes an agent that alters the composition of theliquid as it contacts the second portion. In various embodiments of anyof the aspects delineated herein, the second portion comprises a liquidpermeable material that acts as a filter to remove particulates. Invarious embodiments of any of the aspects delineated herein, the secondportion comprises an agent (e.g., a buffer, a surfactant, or a salt)that alters a physical characteristic (e.g., pH, salt concentration, orbuffering capacity of the liquid sample) of a liquid sample flowingthrough the second portion.

In various embodiments of any of the aspects delineated herein, the testdevice further includes a fourth portion that acts as a wick, the fourthportion including sorbent material. In various embodiments of any of theaspects delineated herein, the fourth portion including absorbentmaterial that facilitates the flow of liquid through the device.

In various embodiments of any of the aspects delineated herein, the testdevice or kit is used at a point of care. In various embodiments of anyof the aspects delineated herein, the liquid sample is blood, urine, orsaliva. In various embodiments of any of the aspects delineated herein,the test device further includes a solid support selected from the groupconsisting of a sheet, tube, and stick. In various embodiments of any ofthe aspects delineated herein, the color change is detected by visualinspection. In various embodiments, the color change is compared to astandard for detecting a difference in color of said sample

In various embodiments of any of the aspects delineated herein, the kitincludes one or more of a powdered reaction mix, a reconstitutionbuffer, and dispensing pipette. In various embodiments of any of theaspects delineated herein, the kit includes a standard for detecting adifference in color of said sample In various embodiments of any of theaspects delineated herein, the kit includes instructions for the use ofthe kit or device for the detection of oxalate.

The invention provides compositions and methods suitable for monitoringoxalate levels at a point of care (e.g., at a patients' home, clinic,physician's office, or other clinical setting). Advantageously, methodsof the invention do not require sophisticated laboratory equipment, butcan be carried out by a patient at home, in a physician's office, or atother sites where patient care is carried out. Compositions and articlesdefined by the invention were isolated or otherwise manufactured inconnection with the examples provided below. Other features andadvantages of the invention will be apparent from the detaileddescription, and from the claims.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of

Microbiology and Molecular Biology (2nd ed. 1994); The CambridgeDictionary of Science and Technology (Walker ed., 1988); The Glossary ofGenetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); andHale & Marham, The Harper Collins Dictionary of Biology (1991). As usedherein, the following terms have the meanings ascribed to them below,unless specified otherwise.

By “agent” is meant any biological or synthetic chemical compound.Exemplary agents include salts, detergents, buffers, acids, bases,polypeptides, nucleic acid molecules, or fragments thereof.

By “alteration” is meant a change (increase or decrease) in the levelsor activity of an analyte, gene or polypeptide as detected by standardart known methods such as those described herein. As used herein, analteration includes a detectable change in the level of oxalate. Such achange may be by about 10%, 25%, 50%, 75% or more. Levels of oxalate maybe monitored by detecting, for example, formate or peroxide levels,using a visual readout.

By “alters the composition of the liquid” is meant produces a change ina physical property of the liquid.

By “ameliorate” is meant decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a disease.

By “analyte” is meant any compound under investigation using ananalytical method. In various aspects of the invention, levels of theanalyte oxalate are monitored.

By “analyte-binding conjugate” is meant a detectable molecule that bindsa compound under investigation.

By “biological sample” is meant any tissue, cell, fluid, or othermaterial derived from an organism (e.g., human subject). In variousembodiments, biological samples include, for example, urine, blood,plasma, feces, gastric samples, mucous, and semen. In variousembodiments, biological fluids include, for example, urine, blood,plasma, gastric samples, mucous, saliva, and semen.

By “capillary communication” is meant facilitating the flow of a liquidbetween liquid permeable materials.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “ includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

By “decarboxylase activity” is meant the activity of an enzyme thatcatalyzes the addition or removal of a carboxyl group from a compound(e.g., oxalate). An enzyme having decarboxylase activity used in theinvention is oxalate decarboxylase.

By “dehydrogenase activity” is meant an activity of an enzyme thatoxidizes a substrate by a reduction reaction that transfers one or morehydrides (H⁻) to an electron acceptor (e.g., NAD⁺/NADP⁺ to NADH/NADPH).An enzyme having dehydrogenase activity used in the invention is formatedehydrogenase.

“Detect” refers to identifying the presence, absence or amount of theanalyte to be detected.

By “indicator system” is meant a system that produces a colored ordetectable derivative of a labeled molecule when incubated with a propersubstrate, allowing it to be detected and/or quantified. Indicatorsystems may further comprise one or more enzymes. In particularembodiments of the invention, the indicator system comprises3-methyl-2-benzothiazolinone hydrazone (MBTH), 3-(dimethylamino) benzoicacid (DMAB), and peroxidase for the detection of hydrogen peroxide(e.g., produced by oxalate oxidase). In other embodiments of theinvention, the indicator system comprises phenazine methosulfate (PMS),iononitro tetrazolium (INT), NAD, and formate dehydrogenase for thedetection of formate (e.g., produced by oxalate decarboxylase).

By “lateral flow device” is meant a test device that relies on the flowof a liquid via capillary action, wicking, or wetting a liquid permeablemedia present in the device.

By “liquid permeable material” is meant a material susceptible towetting, wicking, or transport of a liquid by capillary action.

By “marker” is meant a marker having an alteration in level that isassociated with a disease or disorder.

By “oxidase activity” is meant the activity of an enzyme that catalyzesan oxidation-reduction reaction involving molecular oxygen (O₂) as theelectron acceptor (e.g., reducing oxygen is reduced to hydrogen peroxide(H₂O₂)). An enzyme having oxidase activity used in the invention isoxalate oxidase.

By “peroxidative activity” is meant the activity of an enzyme thatcatalyzes a reaction of the form: ROOR′+electron donor (2e−)+2H+→ROH+ROH. An enzyme having peroxidative activity used in theinvention is horseradish peroxidase.

By “portion” is meant some fraction of a whole. A portion of a testdevice, for example, may be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9of the length of the interior flow path of the device.

By “product” or “reaction product” is meant a compound produced by thereaction of one or more compounds reaction, e.g., in the presence of anenzyme. For example, hydrogen peroxide and carbon dioxide are reactionproducts of the reaction of oxalate and oxygen in the presence ofoxalate oxidase.

By “reduces” is meant a negative alteration of at least 10%, 25%, 50%,75%, or 100%.

By “reference” is meant a standard or control condition.

By “sample application site” is meant the portion of the device thatcontacts a liquid under analysis.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

By “test device” is meant a device used in the detection of an analytein a sample.

By “wick” is meant sorb (e.g., absorb, adsorb) a liquid.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a”, “an”, and “the” areunderstood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an embodiment of a strip that includes reagent pad 11 sothat a sample can be applied to one side of the reagent pad 11 whilelight reflectance is measured from the side of the reagent pad 11opposite the location where sample is applied.

FIG. 2 shows a system in which the reagent is applied to the side withthe hole 14 in the backing handle 12 while light is reflected andmeasured on the other side of the reagent pad 11.

FIGS. 3A-3C depict assays to test for oxalate using test device stripsimbedded with oxalate oxidase enzyme. FIG. 3A depicts the detection ofoxalate in solution using test device strips imbedded with oxalateoxidase enzyme. Test device strips were placed into solutions containingthe indicated mM concentrations of oxalate for 45 seconds. FIG. 3Bdepicts no reaction of oxalate solutions in the absence of oxalateoxidase. As a control, test device strips lacking oxalate oxidase wereplaced into solutions containing the indicated mM concentrations ofoxalate for 45 seconds. FIG. 3C depicts color development in oxalatesolutions exposed to test strips imbedded with oxalate oxidase for 1minute.

FIGS. 4A-4G show the enzyme activity of specific quantities of theoxalate oxidase enzyme in a constant amount of oxalate, thusrepresenting a dilution curve of the enzyme. FIG. 4A depicts thedetection of 0.6M oxalate in solution after 1 min. using test devicestrips imbedded with oxalate oxidase enzyme (0.95-15 IU). FIG. 4Bdepicts the detection of 0.6M oxalate in solution after 5 min. usingtest device strips imbedded with oxalate oxidase enzyme (0.95-15 IU).FIG. 4C depicts the detection of 0.6M oxalate in solution after 5 min.using test device strips imbedded with oxalate oxidase enzyme (0.95-15IU). FIG. 4D depicts color development in an oxalate solution (0.6M)exposed to test strips imbedded with oxalate oxidase (0.95-15 IU) for 5min. FIG. 4E depicts color development in an oxalate solution (0.6M)exposed to test strips imbedded with oxalate oxidase (0.95-15 IU) for 10min. FIG. 4F depicts color development in an oxalate solution (0.6M)exposed to test strips imbedded with oxalate oxidase (0.95-15 IU) for 5min. FIG. 4G depicts color development in an oxalate solution (1.25 M)exposed to test strips imbedded with oxalate oxidase (0.95-15 IU) for 3min.

DETAILED DESCRIPTION OF THE INVENTION

The invention features compositions and methods that are useful formonitoring oxalate levels in a biological sample of a subject. Currentmethods for monitoring oxalate levels are performed in diagnosticlaboratories and require twenty-four hour urine collections.Advantageously, the present invention provides for the monitoring ofoxalate levels in fresh urine or blood samples at a point of care (e.g.,patient's home, physician's office).

Oxalate Detection

Oxalate was confirmed as a normal constituent of urine in 1951, but onlyrecently has the significance of calcium oxalate crystalluria and itsrelationship to urinary tract stone formation been fully recognized.Formation of the sparingly soluble calcium salt of oxalate in theurinary tract is considered a major factor in urolithiasis. In theurine, oxalate is a very strong promoter of calcium oxalateprecipitation.

Oxalate present in a biological sample can be easily detected byoxidizing oxalate to form hydrogen peroxide. Hydrogen peroxide is thendetected as a readout of oxalate levels using any method known in theart.

Enzymes for converting oxalate to hydrogen peroxide include oxalateoxidase. Optimum oxalate oxidase concentration depends on thecomposition of the indicator system. Oxalate oxidase activity in therange from about 500-10,000 U/mL is generally suitable, preferably fromabout 700-2000 U/mL. Generally, higher concentrations of oxalate oxidasecause the reaction to proceed more rapidly and lower concentrations,less rapidly. Optimum concentration can be determined by routineexperimentation.

In one exemplary reaction, oxalate oxidase catalyzes the oxidation ofoxalate to carbon dioxide and peroxide (H₂O₂). The hydrogen peroxidereacts with 3-methyl-2-benzothiazolinone hydrazone (MBTH) and3-(dimethylamino) benzoic acid (DMAB) in the presence of peroxidase toyield an indamine dye, which has an absorbance maximum of 590 nm. Theintensity of the color produced is directly proportional to theconcentration of oxalate in the sample.

In a specific embodiment, the hydrogen peroxide so produced reacts withthe component for detecting hydrogen peroxide, which comprises, forexample, a peroxidase that selectively catalyzes a reaction between thehydrogen peroxide and an indicator. The peroxidase uses hydrogenperoxide as an oxidant which is capable of removing hydrogen atoms fromvarious substrates. A suitable peroxidase may containferriprotoporphyrin, a red hemin obtained from plants. Peroxidasesobtained from animals, for example from the thyroid glands of animals,are also suitable. Horseradish peroxidase (HRPO) is one example of aconstituent of the component for detecting hydrogen peroxide.Chromogenic substrates of peroxidase include, without limitation,N,N-Dimethyl-1,4-phenylene-diamine Dihydrochloride, 1-Naphthol,3-Amino-9-ethylcarbazole, 4-Chloro-1-naphthol, 3,3′-Diaminobenzidine,3,3′-Diaminobenzidine Tetrahydrochloride Hydrate, o-DianisidineDihydrochloride, o-Dianisidine, 4-Aminoantipyrine Hydrochloride,4-Aminoantipyrine, 5-Aminosalicylic Acid, 2,4-Dichlorophenol,N,N-Dimethylaniline N,N-Diethyl-m-toluidine,2,4,6-Tribromo-3-hydroxybenzoic Acid,2,2′-Azinobis(3-ethylbenzo-thiazoline-6-sulfonic Acid Ammonium Salt),1,2-Phenylenediamine, and 3,3′,5,5′-Tetramethylbenzidine. The hydrogenperoxide, preferably catalyzed by a peroxidase, reacts either directlyor indirectly with a substrate to form an indicator dye. The indicatordye may reduce reflectance at the testing surface. Testing surfacereflectance is typically measured at two wavelengths (e.g., about 590 nmand about 700 nm).

The following patents are cited and incorporated by reference forgeneral background of devices that utilize reflectance produced frombreakdown of an analyte and production of hydrogen peroxide. U.S. Pat.No. 4,935,346, issued Jun. 19, 1990 to R. Phillips et al., discloses ameter, strip, and method for determining the glucose concentration in asample of whole blood (see also U.S. Pat. No. 5,304,468). The methodinvolves simply applying a sample of whole blood to a first (“sample”)surface of an inert porous matrix that is impregnated with a reagent.The sample migrates toward the opposite, “testing” surface, as theanalyte glucose interacts with the reagent to produce a light-absorbingreaction product. A reading of reflectance from the testing surfaceindicates the glucose concentration. Reflectance measurements are madeat two separate wavelengths in order to eliminate interferences. Atiming circuit is triggered by an initial decrease in reflectance causedby wetting of the testing surface by the sample having passed throughthe matrix. U.S. Pat No. 5,306,623, issued Apr. 26, 1994 to Kiser etal., discloses a visual blood glucose test strip that involves applyinga glucose-containing whole blood sample to one side of the strip andtaking the glucose reading on the opposite side, after red blood cellshave been separated out and the sample has reacted with a reagent in thestrip. An anisotropic polysulfone membrane was found especially usefulas a single layer matrix for the strip. U.S. Pat No. 5,453,360, issuedSep. 26, 1995 to Y. S. Yu, discloses a dye couple useful in dry reagentstrips for detecting analytes in biological fluids. The dye couplecomprises 3-methyl-2-benzothiazolinone hydrazone and8-anilino-1-naphthalenesulfonate and is used as an indicator in areaction cascade producing a strong oxidizing agent, such as hydrogenperoxide. An advantage of the couple is that it is soluble in aqueoussolution, but becomes insoluble upon oxidative coupling, therebyminimizing fading and providing a stable endpoint.

A meter that has come into widespread use for self-monitoring of bloodglucose is the One Touch® II meter, which uses a strip that is describedin U.S. Pat. Nos. 4,935,346 and 5,304,468, discussed above. The meterand strip permit a user to measure glucose concentration in a wholeblood sample quickly, easily, and accurately. The sample is applied toone surface of the strip and the measurement made on the oppositesurface. A portion of the whole blood sample penetrates from the samplesurface to the testing surface, and the blood color can be observed fromthe testing surface. Certain embodiments of the present invention can beadapted for use with traditional glucose meters such as one working onprinciples similar to the One Touch® meter. In a specific embodiment,the invention pertains to a system for detecting oxalate that comprisesa strip or similar substrate having oxalate oxidase embedded thereon.The strip is contacted with a biological sample such as blood or urineand any oxalate in the specimen reacts with the oxalate oxidase on thestrip to produce hydrogen peroxide. The strip also has embedded thereona component for detecting hydrogen peroxide. When hydrogen peroxide isproduced the component for detecting hydrogen peroxide causes areflectance change that can be detected by a suitable instrument.

Oxalate decarboxylase is a manganese dependent enzyme that catalyzes theconversion of oxalate to formate and carbon dioxide. In anotherexemplary reaction, oxalate decarboxylase catalyzes the decarboxylationof oxalate to formate and CO₂. Formate dehydrogenase is then oxidized toCO₂ in the presence of β-nicotinamide adenine dinucleotide.

The formate can then be detected using a variety of techniques known inthe art. In a preferred embodiment, the production of formate ismeasured colorimetrically by linking the catabolism of formate with theproduction of a detectable color change (e.g., the formation of acompound that absorbs a particular wavelength of light). For example,formate in the presence of the co-factor NAD and the enzyme formatedehydrogenase reduces NAD to NADH. NADH in the presence of phenazinemethosulfate (PMS) forms an intermediate that in the presence of atetrazolium salt (e.g., iononitrotetrazolium (INT) or other tetrazoliumcompounds) produces a color change of the dye. The use ofiononitrotetrazolium (INT) causes a solution containing oxalate to turnblue. The production of formate is directly correlated with the amountof oxalate present in the sample. Therefore, if a known amount offormate is produced using the subject enzyme system, then the amount ofoxalate present in the sample can be easily quantitated. The assaysystems described herein may also be used to identify or screen foroxalate oxidase and oxalate decarboxylase activity in a sample.

Diagnostics

The present invention provides methods of treating disease and/ordisorders or symptoms thereof which comprise administering atherapeutically effective amount of a pharmaceutical compositioncomprising a compound of the formulae herein to a subject (e.g., amammal such as a human). In one embodiment, the invention provides ameans of identifying a patient in need of treatment and/or monitoringtreatment progress. The method includes the step of determining a levelof diagnostic marker (Marker, e.g., oxalate). Thus, one embodiment is amethod of treating a subject suffering from or susceptible to an oxalaterelated disease or disorder or symptom thereof (e.g., kidney stoneformation, vulvodynia, hyperoxaluria type 1, autism, intestinalresections, among others). The method includes the step of administeringto the mammal a therapeutic amount of an amount of a compound hereinsufficient to treat the disease or disorder or symptom thereof, underconditions such that the disease or disorder is treated. For elevatedoxalate urine levels, one treatment is to increase water or fluid intaketo increase voided volumes.

In one embodiment, the invention provides a method of monitoringtreatment progress. The method includes the step of determining a levelof diagnostic marker (Marker) (e.g., any target delineated hereinmodulated by a compound herein, a protein or indicator thereof, etc.) ordiagnostic measurement (e.g., screen, assay) in a subject suffering fromor susceptible to a disorder or symptoms thereof associated withelevated oxalate levels, in which the subject has been administered atherapeutic amount of a compound herein sufficient to treat the diseaseor symptoms thereof. The level of Marker determined in the method can becompared to known levels of Marker in either healthy normal controls orin other afflicted patients to establish the subject's disease status.In preferred embodiments, a second level of Marker in the subject isdetermined at a time point later than the determination of the firstlevel, and the two levels are compared to monitor the course of diseaseor the efficacy of the therapy. In certain preferred embodiments, apre-treatment level of Marker in the subject is determined prior tobeginning treatment according to this invention; this pre-treatmentlevel of Marker can then be compared to the level of Marker in thesubject after the treatment commences, to determine the efficacy of thetreatment.

Design of a Test Device

The test device can take any form desired that provides for the flow ofa liquid test sample from the point of contact with the test sample pastthe test and/or control sites. In general, the test device of thepresent invention includes an interior flow pathway that includes one ormore liquid permeable materials. In one example, the solid substrate isa hydrophilic porous matrix to which reagents may be covalently ornon-covalently bound. The matrix allows for the flow of an aqueousmedium through it. It also allows for binding of protein compositions tothe matrix without significantly adversely affecting the biologicalactivity of the protein, e.g. enzymatic activity of an enzyme. To theextent that proteins are to be covalently bound, the matrix will haveactive sites for covalent bonding or may be activated by means known tothe art. The composition of the matrix is reflective, and it hassufficient thickness to permit the formation of a light absorbing dye inthe void volume or on the surface to substantially affect thereflectance from the matrix. The matrix may be of a uniform compositionor a coating on a substrate providing the necessary structure andphysical properties, such as hydrophilicity. Polysulfones and polyamides(nylons) are examples of suitable matrix materials. Other polymershaving comparable properties may also be used. The polymers may bemodified to introduce other functional groups which provide for chargedstructures, so that the surfaces of the matrix may be neutral, positive,or negative.

In a first portion, the device includes a site for the application of aliquid sample.

The second portion of the device has a variety of features that enhancefunctionality. In one embodiment, the second portion is composed of amaterial capable of filtering the sample to prevent the flow ofparticulate matter through the device. In another embodiment, the secondportion facilitates complex formation by increasing the time requiredfor the liquid to flow from the site of application to the test site.Accordingly, the dimensions of the second portion may be altered (e.g.,increased or decreased) to empirically determine for each applicationthose dimensions that enhance sensitivity while reducing falsepositives, i.e., optimizing the signal-to-noise ratio. In yet anotherembodiment, the second portion of the device can be used to deliver adesired agent to the liquid as it flows through the device. For example,the second portion may be impregnated with a buffer (e.g., TRIS, sodiumcarbonate), surfactant (e.g., Tween, Triton), preservative (e.g., Naazide, thimerosol), salt, or other agent, such that contact of thesample with the second portion of the device alters the sample.Exemplary alterations include an increase or decrease in the pH of thesample, in the salt concentration, in the buffering capacity, or in thebinding between the conjugate and the analyte.

The third portion of the device includes a test site, which acts as areadout zone that provides for detection of oxalate in the sample.Various means for detecting the presence of an analyte at a test siteare known in the art. In various embodiments, an enzyme system havingoxalate specific enzyme activity is fixed to the medium at the testsite, the enzyme system comprising an oxalate specific enzyme, asubstance which reacts with a metabolite of the oxalate specific enzyme,and an indicator material which reacts in the presence of the metaboliteand changes color. Oxalate specific enzyme activity in the range fromabout 500-10,000 U/mL is generally suitable, preferably from about700-2000 U/mL. Optimum oxalate specific enzyme concentration depends onthe composition of the indicator system, and can be determined byroutine experimentation. In some embodiments, the oxalate specificenzyme is oxalate oxidase (e.g., a purified and/or recombinant oxalateoxidase). In other embodiments, the oxalate specific enzyme is oxalatedecarboxylase (e.g., a purified and/or recombinant oxalatedecarboxylase). In this approach, detection of color or a color changeindicates the presence of oxalate in the sample.

It may be desirable to include a positive control to indicate that theliquid sample has traversed the interior flow path from the site ofapplication past the test site.

The device may also include in a fourth portion a wicking pad thatcontains sorbent material capable of absorbing or adsorbing excessliquid present in the liquid sample.

In one embodiment, the test device contains a liquid permeable materialdefining the following portions in capillary communication:

a) a first portion that is the site for application of a liquid sample,comprising a liquid permeable medium, where the first portion is between5 mm and 20 mm in length; for example, the length of the first portionis equal to any integer between 5 and 20 (5, 10, 15, 20 mm in length);

b) a second portion comprising a liquid permeable medium, where thesecond portion overlaps the first portion by at least 1, 2, 3, 4, or 5mm; and the length of the second portion is between 10 mm and 40 mm; forexample, the length of the second portion is any integer between 10 and40 (e.g., 10, 15, 20, 25, 30, 35, 40); and

c) a third portion that is the site for detecting the presence ofoxalate at the test site, the third portion comprising a liquidpermeable medium having an enzyme system having oxalate specific enzymeactivity fixed to the medium at the test site, the enzyme systemcomprising oxalate specific enzyme, a substance which reacts with ametabolite of the oxalate specific enzyme, and an indicator materialwhich reacts in the presence of the metabolite and changes color,wherein the third portion is between 15 and 40 mm in length; forexample, is any integer between 15 and 40 (e.g., 15, 20, 25, 30, 35,40); and the second portion overlaps the third portion by at least 1, 2,3, 4, or 5 mm

In a fourth portion the device contains sorbent material. The sorbentmaterial has a length between 25 and 75 mm For example, the length is aninteger between 25 and 75 (e.g., 25, 35, 50, 60, 70, 75). In oneembodiment, the fourth portion overlaps the third portion by at least 1,2, 3, 4, or 5 mm

In general the interior flow path is between 1 mm and 10 mm in width;for example, the width of a test device (e.g., test strip) is anyinteger between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10). In oneembodiment the width of the strip is 3.8 mm The design and dimensions ofan exemplary test strip of the invention are shown in FIG. 1. Desirably,a test device of the invention has increased sensitivity relative to aconventional test device. Sensitivity of a test device of the inventionis increased by at least 5%, 10%, 25%, 50%, 75%, 100%, 150%, or 200%relative to a conventional test device.

A schematic diagram showing an exemplary test device is provided at FIG.1, which shows an embodiment of a strip that includes reagent pad 11 sothat a sample can be applied to one side of the reagent pad 11 whilelight reflectance is measured from the side of the reagent pad 11opposite the location where sample is applied.

FIG. 2 shows a system in which the reagent is applied to the side withthe hole 14 in the backing handle 12 while light is reflected andmeasured on the other side of the reagent pad 11. Other structures thanthe one depicted may be employed. The pad 11 may take various shapes andforms, subject to the limitations provided herein. The pad 11 will beaccessible on at least one surface and usually two surfaces. A reagentelement may be attached to the support by any convenient means, e.g., aholder, clamp or adhesives; however, in the illustrated embodiment it isbonded to the backing. Methods of bonding include, but are not limitedto, use of any non-reactive adhesive, by a thermal method in which thebacking surface is melted enough to entrap some of the material used forthe reagent element, or by microwave or ultrasonic bonding methods whichlikewise fuse the hydrophilic sample pads to the backing. It isimportant that the bonding the such as to not itself interferesubstantially with the diffuse reflectance measurements or the reactionbeing measured, although this is unlikely to occur as no adhesive needbe present at the location where the reading is taken. For example, anadhesive 13 can be applied to the backing strip 12 followed first bypunching hole 14 into the combined strip and adhesive and then applyingreagent pad 11 to the adhesive in the vicinity of hole 14 so that theperipheral portion of the reagent pad attaches to the backing strip.

Interior Flow Path

The test device comprises an interior flow path that facilitates theflow of a liquid sample through the device. This interior flow pathcontains one or more liquid permeable materials or membranes composed ofany relatively inert material or a combination of materials suitable fortransporting a liquid (e.g., glass fibers, polyester, nitrocellulose,fibers of cellulose or derivatives thereof, non-cellulose hydrocarbonmaterials, ceramics) from the contact site past the test and/or controlsites and, optionally, into a reservoir. Suitable materials for use inthe interior flow path are wettable and exhibit low non-specificbinding. Materials having increased sorptivity promote the flow ofliquid. Different materials having different absorption characteristicsor sorptivities may be used in various portions of the flow path. Ifdesired, the materials to be used are screened for optimal pore size anddensity in order to facilitate the controlled distribution of anantibody within a membrane, to optimize reaction kinetics, or tooptimize the sensitivity, discriminatory ability, or signal-to-noiseratio of the device.

Solid Supports

For most applications, the test device includes an interior flow pathwayfixed to a solid support. The physical shape of the solid support is notcritical, although some shapes may be more convenient than others forthe present purpose. Accordingly, the solid support may be in the shapeof a paper strip, dipstick, membrane (e.g. a nylon membrane or acellulose filter), a plate (e.g. a microtiter plate) or solid particles(e.g. latex beads). The solid support may be made of any suitablematerial, including but not limited to a plastic (e.g., polyethylene,polypropylene, polystyrene, latex, polyvinylchloride, polyurethane,polyacrylamide, polyvinylalcohol, nylon, polyvinyl acetate, or anysuitable copolymers thereof), cellulose (e.g. various types of paper,such as nitrocellulose paper and the like), a silicon polymer (e.g.siloxane), a polysaccharide (e.g. agarose or dextran), or an ionexchange resin (e.g. conventional anion or cation exchange resins).

Sorbent Reservoir

The test device optionally includes a fourth portion that forms areservoir of adsorbent or absorbent material. This reservoir sorbsexcess liquid as it flows through the test device. For someapplications, such as where the concentration of oxalate is particularlylow, it may be desirable to apply large volumes of a liquid test sampleto the test device. In such cases, the presence of the adsorbentmaterial may enhance the sensitivity of oxalate detection. Optionally,the region of the flow path in the test cell defining the test andcontrol sites is restricted in cross-sectional area relative to otherregions of the flow path. This feature produces a “bottle-neck” effectwherein the oxalate in the entire volume of adsorbed sample must passthrough an area of restricted flow immediately above the test site. This“bottle-neck” may facilitate sandwich formation. Suitable sorbentmaterials include virtually any commercial material (e.g., synthetic ornatural materials, such as cotton) capable of absorbing many times itsweight in water. Such materials are widely available in commerce.

Capture Molecule

The test device optionally includes a portion that contains a capturemolecule. In a specific embodiment, the oxalate capture molecule is acalcium binding protein. Depending on the sample and level of capturedesired, the calcium binding proteins, or other oxalate capturemolecules used, may be pretreated with calcium thereby resulting inloaded calcium proteins. In a further embodiment, the calcium bindingproteins are attached to the substrate. In a more specific embodiment,the calcium binding proteins are attached to the substrate by linkermolecules. In an alternative embodiment, the calcium binding proteinsare embedded in the substrate. In a further embodiment, the inventionpertains to a micro-particle linked to an oxalate capture molecule(e.g., calcium ions which bind oxalate in the biological sample). Theparticle is associated with a thiol group which is bound to a linkermolecule such as PEG. Those skilled in the art will appreciate thatother linker molecules known in the art could be used in accordance withthe teachings herein. The linker is associated with an oxalate capturemolecule. The microparticles can be applied to a solid substrate. In apreferred embodiment, the microparticles are printed onto a substrate.

Methods of Using the Test Device

The invention provides methods of using a test device of the inventionfor the detection of oxalate in a test sample. In one example, the assayis conducted by placing the leading edge (first portion) of a lateralflow device in contact with a liquid test sample. In another example,the sample is brought into contact with the device by applying a liquidtest sample to the first portion of the lateral flow device in adrop-wise fashion.

Test Samples

Methods and compositions of the invention are useful for theidentification of oxalate in a test sample. In one embodiment, themethods of the invention are suitable for detecting analytes ofbiological origin. Test samples include, but are not limited to, anyliquid containing a dissolved or dispersed analyte of biological origin.Exemplary test samples include body fluids (e.g. blood, serum, plasma,amniotic fluid, sputum, urine, cerebrospinal fluid, lymph, tear fluid,feces, or gastric fluid), or feces. If the test sample is not in itselfsufficiently fluid for the present purpose, it may be admixed with asuitable fluid to the desired fluidity.

Kits

The invention provides kits that include a composition or a test devicefor the detection of oxalate in a sample. In one embodiment, the kitincludes a powdered reaction mix and a reconstituting reaction bufferdescribed herein. In some embodiments, the kit comprises a container,which contains the powdered reaction mix and another container, whichcontains reconstituting reaction buffer; such containers can be boxes,ampoules, bottles, vials, tubes, bags, pouches, blister packs, or othersuitable container forms known in the art. In one embodiment, suchcontainers may be sterile. Such containers can be made of plastic,glass, laminated paper, metal foil, or other materials suitable forholding medicaments. The powder formulation may include an oxalatespecific enzyme composition to catalyze and detect oxalate in thebiological sample after exposing said substrate to a biological samplecontaining oxalate, and a chromogenic reactive substrate for detectingwhether the sample contains oxalate. This method provides for rapid,inexpensive, easy to use testing for oxalate in a sample. A color changemay be detected as a difference in the hue or shade of color. The changein color may be indicative of either the presence or the quantity ofoxalate in a sample. In one embodiment, a color chart for visuallyscoring the presence of oxalate in the sample is included.

In one embodiment, the kit includes a lateral flow device describedherein. In some embodiments, the kit comprises a container, whichcontains the lateral flow device; such containers can be boxes,ampoules, bottles, vials, tubes, bags, pouches, blister packs, or othersuitable container forms known in the art. In one embodiment, suchcontainers may be sterile. Such containers can be made of plastic,glass, laminated paper, metal foil, or other materials suitable forholding medicaments.

If desired, the composition or device is provided together withinstructions for using it to identify the presence or absence of oxalatein a sample. The instructions will generally include information aboutthe use of the device for the identification of a particular analyte,such as oxalate in a liquid sample (e.g., an environmental sample,biological sample, or liquid sample extracted from an agriculturalcommodity). The instructions may be printed directly on the container(when present), or as a label applied to the container, or as a separatesheet, pamphlet, card, or folder supplied in or with the container. Ifdesired, the kit may also include a color chart. The color chartprovides a means of indicating the oxalate levels in the sample base onthe color reaction. If desired, the kit may also include a standardmeasure pipet, a test vial, and/or a liquid (e.g., suitable buffer, suchas phosphate buffered saline, or water) to be used in the extraction ofa sample.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook,1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture”(Freshney, 1987); “Methods in Enzymology” “Handbook of ExperimentalImmunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells”(Miller and Calos, 1987); “Current Protocols in Molecular Biology”(Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques areapplicable to the production of the polynucleotides and polypeptides ofthe invention, and, as such, may be considered in making and practicingthe invention. Particularly useful techniques for particular embodimentswill be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the assay, screening, and therapeutic methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention.

EXAMPLES Example 1 Oxalate Test Strips Imbedded with Oxalate OxidaseEnzyme Detected Oxalate in Solution

Oxalate test strips were imbedded with oxalate oxidase enzyme. Teststrips were placed into solutions containing known concentrations ofoxalate for 45 seconds. Oxalate was detected in solution using the teststrips (FIG. 3A). The sensitivity of the strips was determined to bebetween 0.62 and 0.31 mM oxalate. As a control, test device stripslacking oxalate oxidase were placed into solutions containing knownconcentrations of oxalate. No color reaction was detected for theoxalate solutions in the absence of oxalate oxidase (FIG. 3B). Solutionsof oxalate were exposed to test strips imbedded with oxalate oxidase for1 minute. Color development was observed in oxalate solutions exposed totest strips imbedded with oxalate oxidase for 1 minute (FIG. 3C). Basedon the differential between 0.62 and 0.31 mM oxalate solutions, thesensitivity was determined to be between 0.62 and 0.31 mM oxalate. Anoxalate concentration >0.5 mM is indicative of hyperoxaluria.

Enzyme activity of specific quantities of the oxalate oxidase enzyme ina constant amount of oxalate were tested. Oxalate test strips wereimbedded with varying amounts of oxalate oxidase enzyme. As the amountof oxalate is held constant while the amount of oxalate oxidase isvaried, the reactions represent a dilution curve of the enzyme.

Oxalate test strips were imbedded with 0.95-15 IU oxalate oxidaseenzyme/strip. The strips were used to test an oxalate solution (0.62 mMoxalate) and the reactions observed at 1 min. and 5 min. time points. Atthese time points, oxalate test strips having at least 3.8 IU oxalateoxidase detected oxalate (0.62 mM) in solution (FIGS. 2A-2C). Colordevelopment was also observed in the oxalate solution exposed to thetest strips after 5 min. and 10 min. At these time points, oxalate teststrips having at least 1.9 IU oxalate oxidase changed color, thus,detecting oxalate (0.62 mM) in solution. (FIGS. 4D and 4E). Thus, inthis study oxalate strips having at least 1.9 IU oxalate oxidase areuseful for detecting oxalate. Once the activity of an enzyme preparationis determined, the amount of enzyme preparation to be applied to eachtest strip is determined

Reactions with the test strips on 0.6 mM and 1.25 mM oxalate solutionswere compared. Consistent with other results, oxalate test strips havingat least 1.9 IU oxalate oxidase detected 0.62 mM oxalate in solution, asshown by color development after about 5 min. (FIG. 4F). Oxalate teststrips having at least 0.95 IU oxalate oxidase detected 1.25 mM oxalatein solution after about 3 min. (FIG. 4G).

Thus, oxalate test strips imbedded with oxalate oxidase enzyme provide arapid, inexpensive, sensitive, and highly accurate assay for detectingoxalate that can be used at point of care (e.g., at home).

Example 2 Point-of-Care Oxalate Test Kit

The invention provides a point-of-care test kit for monitoring oxalatelevels in a biological sample (e.g., urine). In certain embodiments, thecontents of the oxalate test kit contains one or more reaction vials,one or more plastic squeeze droppers (e.g., two); one or more plasticbottles of sterile water; and an instruction sheet, including a colorchart. The reaction vials contain an oxalate enzyme system in powderform that produces a color change for monitoring oxalate in solution.The powder formulation comprises oxalate oxalase enzyme (5.0 IUactivity), EDTA (0.0010 mM), DMAB (0.0050 mM), and MBTH (0.0005 mM).Depending on the number of required measurements, the kit may containmore than one of each of the above items. Optionally, the kit containsurine specimen collection cups.

At time of testing for oxalate levels in a urine sample, 1.0 ml of H₂Ois dispensed into a single reaction vial using the dropper provided. Bysqueezing the dropper, water is added up to an indicated marker line.The powder is dissolved by gentle shaking. This solution should beactive for 1 hr. A mid-stream urine specimen is collected. Using adropper provided, 1.0 ml of urine is dispensed into the reaction vial,tightly capped, and shaken. After allowing the the vial sit for 3-5minutes, the color of the solution is compared to the color chartprovided. A reading less than 0.50 mg/ml is considered normal for bothmale and females.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

1. A test device for detecting oxalate in a liquid sample, the devicecomprising oxalate oxidase, and an indicator system that provides adetectable reaction product in response to hydrogen peroxide production.2. The test device of claim 1, wherein the indicator system comprises3-methyl-2-benzothiazolinone hydrazone (MBTH) and 3-(dimethylamino)benzoic acid (DMAB). 3.-8. (canceled)
 9. A test device for detectingoxalate in a liquid sample, the device comprising a liquid permeablematerial defining the following portions in capillary communication: a)a site for application of a liquid sample, comprising a liquid permeablemedium; b) a second portion comprising a liquid permeable medium; and c)a third portion that is the site for detecting the presence of oxalateat the test site, the third portion comprising a liquid permeable mediumhaving an enzyme system having oxalate specific enzyme activity fixed tothe medium at the test site, the enzyme system comprising oxalatespecific enzyme, a substance which reacts with a reaction product of theoxalate specific enzyme, and an indicator material which reacts in thepresence of the reaction product and changes color.
 10. The test deviceof claim 9, wherein the enzyme system has oxalate oxidase activity, theenzyme system comprising oxalate oxidase, a substance havingperoxidative activity, an indicator material which is oxidized in thepresence of peroxide and changes color. 11.-18. (canceled)
 19. The testdevice of claim 9, wherein the liquid sample is blood, urine, or saliva.20. The test device of claim 9, further comprising a solid supportselected from the group consisting of a sheet, tube, and stick.
 21. Thetest device of claim 9, which is used at a point of care.
 22. A methodfor detecting an analyte in a liquid sample, the method comprising: a)applying a liquid sample to the device of claim; and b) detectingpresence or absence of oxalate at a test site, wherein the presence of adetectable reaction product or color change at the test site identifiesthe presence of oxalate in the sample and the absence of color change atthe test site identifies the absence of the oxalate in the sample. 23.The method of claim 22, wherein the step (b) detects the level ofoxalate in the sample.
 24. The method of claim 22 for detecting oxalatein a liquid sample, the method comprising: a) applying a liquid sampleto the device of claim 1; b) providing for the flow of the liquid fromthe site of application to the test site; and c) detecting presence orabsence of oxalate at a test site, wherein the presence of color changeat the test site identifies the presence of oxalate in the sample andthe absence of color change at the test site identifies the absence ofthe oxalate in the sample.
 25. The method of claim 24, the color changeis detected by visual inspection.
 26. The method of claim 25, furthercomprising comparing the color change to a standard for detecting adifference in color of said sample.
 27. (canceled)
 28. A kit comprisinga reaction mix, the reaction mix comprising an enzyme system havingoxalate specific enzyme activity, the enzyme system comprising oxalatespecific enzyme, a substance which reacts with a reaction product of theoxalate specific enzyme, and an indicator which reacts in the presenceof the reaction product and changes color; and a color chart.
 29. Thekit of claim 28, wherein the enzyme system has oxalate oxidase activity,the enzyme system comprising oxalate oxidase, a substance havingperoxidative activity, an indicator which is oxidized in the presence ofperoxide, and said substance having peroxidative activity and changescolor.
 30. The kit of claim 28, wherein the enzyme system has oxalatedecarboxylase activity, the enzyme system comprising an oxalatedecarboxylase, a substance having formate dehydrogenase activity, and anindicator which is reduced in the presence of formate and changes color.31. The kit of claim 28, comprising one or more of a powdered reactionmix, a reconstitution buffer, and dispensing pipette.
 32. The kit ofclaim 28, further comprising a standard for detecting a difference incolor of said sample
 33. The kit of claim 28, further comprisinginstructions for the use of the kit for the detection of oxalate. 34.The kit of claim 28, which is used at a point of care. 35.-36.(canceled)